Use of anti-cd1 antibodies for the modulation of immune responses

ABSTRACT

The invention provides methods for the administration of an anti-CD 1 antibody for the treatment or prevention of a variety of disorders, such as autoimmune disease, viral infection, bacterial infection, parasitic infection, infection by a eukaryotic pathogen, allergy, asthma, inflammatory condition, graft versus host disease, graft rejection, immunodeficiency disease, spontaneous abortion, pregnancy, and cancer.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was funded by grants AI42955 and CA89567 from theNational Institute of Health. The government may have certain rights inthe invention.

BACKGROUND OF THE INVENTION

Modulation of the immune system is desirable to treat a variety ofdiseases and disorders including, but not limited to, autoimmunediseases, infections, allergies, asthma, inflammatory conditions,spontaneous abortion, pregnancy, graft versus host disease, and cancers.

Antigen presenting cells (“APC;” e.g., CD1-expressing monocytes,macrophages, dendritic cells, and B cells) are required for protectionagainst numerous diseases. APCs modulate immune responses by producingantibodies, cytokines, and growth factors; destroying infected andcancerous cells, and activating T cells.

T cells are lymphocytes that participate in multiple cell-mediatedimmune reactions, such as the recognition and destruction of infected orcancerous cells. Subsets of T cells, such as suppressor, cytotoxic, andhelper T cells, mediate different immunologic functions. Suppressor Tcells are responsible for turning the immune response off after aninfection is cleared. Cytotoxic or “natural killer” T cells destroyinfected or cancerous cells. Helper T cells produce cytokines thatmodulate the activity of cytotoxic T cells and/or antibody-producing Bcells.

A subset of helper T cells, Th1 cells, secrete interleukin-1 (IL-1),IL-2, gamma interferon (INF-γ), and IL-2 which enhance cell-mediatedresponses such as cytotoxic T cell activity and inhibit both Th2 helperT cell activity and humoral immunity mediated by soluble antibodies. Dueto their ability to kill antigen-presenting cells and theircytokine-mediated effector activity, Th1 cells are associated withvigorous delayed-type hypersensitivity reactions. Th2 cells, the othersubset of helper T cells, are thought to inhibit cell-mediated responsesand to enhance the humoral response. Th2 cells secrete IL-4, IL-5, IL-6,IL-9, IL-10, and IL-13 which activate B cell development and antibodyproduction. T cells may also participate in immune deviation responses,such as the suppression of an ongoing immune response which may involvethe secretion of TGF-β or IL-10 cytokines (Sonoda et al, J. Ex. Med.190:1215-1255, 1999; Streilein et al., Hum. Immunol. 52:138-143, 1997;Hong et al, J. Ex. Med. 190, 1197-1200, 1999; Streilein et al., J.Immunol. 158:3557-3560, 1997).

To recognize a particular antigen bound to an antigen-presenting cell,most T cells express a highly specific T cell receptor (TCR) on theircell surface. The chains of the most common T cell receptors are calledα and β. A second T cell receptor, found on a minor subpopulation of Tcells, is composed of γ and δ chains. The genes for the α, β, γ, and δchains of the T cell receptors have organizations similar to that ofantibody genes: there are libraries of V, D, and J regions from whichmembers are joined to form entire genes.

In contrast to most T cell subpopulations, which have diverse sequencesfor their TCR-a chain, invariant T cells have a highly conservedinvariant TCR-α chain, Vα24-JαQ in humans and Vα14-Jα281 in mice, thatpairs preferentially with human Vβ11 or murine Vβ8. These cells areeither CD4⁺CD8⁻ or CD4⁻CD8⁻. This invariant TCR is presumed to enableinvariant T cells to recognize endogenous or pathogen-derived lipidantigens presented by nonpolymorphic MHC class I-like proteins, calledCD1 family members. Humans have four CD1 proteins (CD1a, CD1b, CD1c, andCD1d), but mice have only a duplicated CD1d gene that is highlyhomologous to human CD1d. Human CD1d is expressed at high levels bythymocytes, at lower levels by B cells and monocytes, and by some cellsoutside of the lymphoid and myeloid lineages.

Many invariant T cells are distinguished by expression of several cellsurface proteins otherwise found largely on natural killer (NK) cells,including CD161 (NKR-P1A) in humans, and a cell surface C-type lectin,NKR-P1C (NK1), in mice. This T cell subpopulation, referred to here as“invariant NK T cells,” represents a major fraction of the mature Tcells in thymus, the major T cell subpopulation in murine liver, and upto 5% of splenic T cells in some mouse strains.

Murine and human invariant T cells also produce large amounts of theimmunoregulatory cytokines IL-4 (a Th2 effector) and IFN-γ (a Th1effector) in vivo in response to an anti-CD3 antibody or to CD1d. Thesecytokines allow the cells to participate in both Th2 and Th1 responses.The role of invariant T cells in augmenting the Th2 response, whichappears to be protective in some autoimmune diseases, is furthersupported by the presence of defects in invariant T cells in a number ofhuman and murine models of autoimmune diseases, including type 1diabetes. Thus, alterations in the balance between Th1 and Th2 responsesinduced by invariant T cells may play a role in the development ofautoimmune diseases.

Invariant T cells can also promote rapid Th1 immune responses andanti-tumor responses. Invariant T cells, which comprise a major fractionof the T cells in murine liver, can be stimulated by IL-12 to becomeactive cytotoxic T cells and protect against liver metastases in tumormodels. This conclusion was confirmed genetically through the generationof Jα281 knockout mice, which do not express the invariant Vα14-Jα281TCR. These mice had markedly diminished numbers of invariant T cells andcould not mediate IL-12 induced tumor rejection. Other studies showedthat IL-12 administration no longer induced an early IFN-γ response inthe spleen and liver of CD1d knockout mice, which are invariant T celldeficient. In addition, data from human patients shows fewer invariantNK T cells and reduced Th1-like responses in patients with advancedcancer. The anti-tumor response of activated invariant T cells could bepartially mediated by their CD1d specific cytotoxicity and NK/LAKcell-like toxicity. Other regulatory functions of invariant T cells,possibly through cytokine production or interactions with antigenpresenting cells (APCs), may also play important roles in anti-tumorimmune responses.

Invariant T cells may also have a role in the pathogenesis ofspontaneous abortion. Stimulation of decidual invariant T cells in miceby administration of a ligand for invariant T cells provoked abortion inpregnant mice. The perforin-dependent killing and production of IFN-γand tumor necrosis factor-α by the invariant T cells were required forthis induction of abortion.

In contrast to human peripheral blood in which invariant T cells are themajor CD1d-reactive subpopulation, human and mouse bone marrow and humanliver have T cell populations dominated by CD1d-reactive noninvariant Tcells using diverse TCRs which can also produce a large amount of IL-4and IFN-γ. These CD1d-reactive noninvariant T cells can be either NK ornon-NK T cells, and they function similarly to CD1d-reactive invariant Tcells. The CD1d-reactive noninvariant T cells in bone marrow may have arole in suppressing graft versus host disease, and both populations mayenhance graft versus leukemia responses. In the liver, these T cells mayprotect against infections, such as Hepatitis C infections, but may alsocause damage due to their Th1 response. Additionally, we found thatCD1d-reactive NK T cells are critical for immune tolerance to antigensin the anterior chamber of the eye, an immune privileged site (Sonoda etal., supra). Such mechanisms may also be important in the maintenance ofperipheral tolerance.

Parasitic glycosyl-phosphatidylinositols derived from Plasmodium,Trypanosoma, or Leishmania have been recently shown to stimulate murineCD1d-reactive invariant Vα14 NK T cells. In addition, anα-galactosylceramide (α-GalCer) lipid, which was isolated from marinesponge in a screen for anti-tumor activity, is a CD1d-presented antigen.α-GalCer is an example of an agent which can be used to expand humanCD1d-reactive invariant T cells from umbilical cord or peripheral bloodsamples that are first enriched for invariant T cells by purificationusing an anti-Vα24 antibody. The enriched Vα24⁺ cells are cocultured inthe presence of α-GalCer and purified antigen-presenting cells (APCs).However, it would be desirable in a clinical setting to use a simplermethod for enhancing protective immune responses that does not requirethe modulation of CD1-reactive T cells.

Thus, there exists a need to specifically modulate the immune system forthe prevention and treatment of diseases and disorders such as diabetes,autoimmune diseases, infections, allergies, asthma, inflammatoryconditions, spontaneous abortion, pregnancy, graft versus host disease,and cancers.

SUMMARY OF THE INVENTION

We have developed novel methods for the prevention, stabilization, ortreatment of a variety of diseases including infectious and autoimmunediseases and cancers. These methods involve administering one or moreanti-CD1 antibodies to a mammal in an amount sufficient to modulate theactivity or number of antigen-presenting cells (APCs).

Accordingly, in a first aspect, the invention provides a method ofpreventing, stabilizing, or treating an autoimmune disease, viralinfection, bacterial infection, parasitic infection, infection by aeukaryotic pathogen, allergy, asthma, inflammatory condition, graftversus host disease, graft rejection, immunodeficiency disease,spontaneous abortion, pregnancy, or cancer in a mammal (e.g., anon-rodent mammal or a human). This method involves administering to themammal one or more anti-CD1 antibodies or antibody fragments in anamount sufficient to prevent, stabilize, or treat the condition. Invarious embodiments, the method increases the activity or number of oneor more APCs by at least 20, 30, 40, 50, 60, 80, 90, 100, 200, 500, oreven 1,000%. In various embodiments, the anti-CD1 antibody isadministered at a dosage level that allows retention of at least 20, 30,40, 50, 60, 80, 90, or 100% of the activity of CD1-reactive T cells(e.g., CD1d-reactive T cells, NK T cells, invariant T cells, or JαQ⁺ Tcells) in the mammal relative to the corresponding level of T cellactivity in the mammal before administration of the anti-CD1 antibody,or relative to the corresponding level of T cell activity in anuntreated mammal. In some embodiments, the method increases the activityof CD1-reactive T cells by at least 20, 30, 40, 50, 60, 80, 90, 100,200, 500, or even 1,000%. In other embodiments, the method decreases theamount of IL-12, TNF, or IFN-γ. In some embodiments, the methoddecreases the amount of IL-12, TNF, and IFN-γ. Desirably, the decreasein the amount of one or more of these cytokines is at least 10, 20, 30,40, 50, 60, 80, 90, or 95%. In some embodiments, the method increasesthe amount of TGF-β, IL-12, IL-1α, or IL-10. Desirably, the increase inthe amount of this cytokine is at least 10, 20, 30, 40, 50, 60, 80, 90,100, 200, 500, or even 1,000%. In particular embodiments, the methodincreases the amount of TGF-β, IL-10, and/or IL-12. In still otherembodiments, the method decreases the amount of IL-12, TNF, or IFN-γ,and increases the amount of TGF-β and/or IL-10. In some embodiments, thecondition is an autoimmune disease other than lupus or a bacterialinfection other than a listeria infection.

In another aspect, the invention provides a method of preventing,stabilizing, or treating APC pathogenesis in a mammal (e.g., anon-rodent mammal or a human). This method involves administering to themammal one or more anti-CD1 antibodies or antibody fragments in anamount sufficient to reduce the number or activity of at least one APC.In various embodiments, the method decreases the activity or number ofone or more APCs by at least 20, 30, 40, 50, 60, 80, 90, 95 or 100%. Inanother desirable embodiment, the antibody or antibody combination iscovalently linked to a toxin, a radiolabel, or a molecule which targetshost defensive or catabolic processes toward the cells. In one desirableembodiment, one or more cytokines are also administered to the animal.In another embodiment, the APC pathogenesis is an autoimmune pathologysuch as a response to a viral infection, such as a Hepatitis infection,picornarirus infection, polio infection, or coxsacchie infection.

In another related aspect, the invention provides another method ofpreventing, stabilizing, or treating an autoimmune disease, viralinfection, bacterial infection, parasitic infection, infection by aeukaryotic pathogen, allergy, asthma, inflammatory condition, graftversus host disease, graft rejection, immunodeficiency disease,spontaneous abortion, pregnancy, or cancer in a mammal (e.g., anon-rodent mammal or a human). This method involves administering to themammal one or more APCs (e.g., dendritic cells) that have been contactedwith one or more anti-CD1 antibodies or antibody fragments. The APCs areadministered in an amount sufficient to prevent, stabilize, or treat thecondition. In a desirable embodiment, the APCs are isolated from amammal, contacted with an anti-CD1 antibody, optionally purified, andthen re-administered to the same mammal. In yet another desirableembodiment, the antibody or antibody combination is administered to anAPC derived from the mammal in vitro, followed by re-infusion of thecell. Desirably, other agents which may potentiate this activity areadministered in vitro or in vivo. In some embodiments, an anti-CD1antibody is also administered to the mammal.

Anti-CD1 antibodies can also be used to increase the immune response toa vaccine that is administered for the treatment or prevention of aninfection.

Accordingly, in one such aspect, the invention features a method ofpreventing, stabilizing, or treating an infection in a mammal (e.g., anon-rodent mammal or a human). This method involves administering to themammal an antigen from an infectious agent and one or more anti-CD1antibodies or antibody fragments in an amount sufficient to prevent,stabilize, or treat the infection. Exemplary antigens include one ormore proteins, peptides, lipids, carbohydrates, nucleic acids, smallmolecules, or intact infectious agents such as bacteria, viruses, oryeast. Other examples of antigens include any commercially availablevaccine, such as an HIV, ebola, or small pox vaccine. The antigen may beadministered to the mammal before, during, or after the administrationof the anti-CD1 antibody.

In another aspect, the invention features a pharmaceutical compositionthat includes an anti-CD1 antibody and (i) an antigen from an infectiousagent, (ii) a vaccine, or (iii) an adjuvant in any pharmaceuticallyacceptable form, including isomers such as salts, solvates, andpolymorphs thereof. In various embodiments, the composition alsoincludes a pharmaceutically acceptable carrier or diluent and/or anadjuvant. In some embodiments, the antigen is inactivated using standardmethods such as heat or chemical (e.g., formaldehyde) inactivation.Exemplary antigens include proteins, peptides, lipids, carbohydrates,nucleic acids, small molecules, or intact infectious agents such as abacteria, viruses, or yeast.

The invention also features methods for increasing the activity ornumber of APCs in vitro or in vivo. In one such aspect, the inventionprovides a method of increasing the production or secretion of acytokine by an APC. This method involves administering to an APC one ormore anti-CD1 antibodies or antibody fragments in an amount sufficientto increase the production or secretion of a cytokine by the APC. Invarious embodiments, the method increases the secretion of one or moreof the following cytokines by at least 20, 30, 40, 50, 60, 80, 90, 100,200, 500, or even 1,000%: IL-1α, IL-2, IL-4, IL-7, IL-10, IL-12, IL-13,IL-15, IL-18, IFN-α/β, IFN-γ, or GM-CSF. In particular embodiments, thesecretion of IL-12 by dendritic cells increases by at least 50, 60, 80,90, 100, 200, 500, or even 1,000%. The APC may be in vitro or in vivo(e.g., in a mammal such as a human). In some embodiments, the APC is inor from a mammal diagnosed with, or at increased risk for, a disease,disorder, or infection selected from the group consisting of: anautoimmune disease, viral infection, bacterial infection, parasiticinfection, infection by a eukaryotic pathogen, allergy, asthma,inflammatory condition, graft versus host disease, graft rejection,immunodeficiency disease, spontaneous abortion, pregnancy, and cancer.In some embodiments, the autoimmune disease is a disease other thanlupus, or the bacterial infection is an infection other than a listeriainfection.

In desirable embodiments of any aspect of the invention, the antibody orantibody combination is administered to the animal intraarticularly,intralesionally, orally, intramuscularly, intravenously, subcutaneously,or intraperitoneally. In another desirable embodiment, the antibody orantibody combination is administered with a pharmaceutically suitablecarrier. Desirably, the antibody or antibody combination is administeredto the animal in a dose that is less than 20, 15, 10, 5, 4, or 1 mg/kg.In some embodiments, the dose falls within one of the following ranges:0.1 to 10 mg/kg, 0.1 to 0.4 mg/kg, 0.1 to 1 mg/kg, or 1 to 4 mg/kg,inclusive.

In various embodiments of any aspect of the invention, the anti-CD1antibody is administered at a dosage level that allows retention of atleast 20, 30, 40, 50, 60, 80, 90, or 100% of the activity ofCD1-reactive T cells (e.g., CD1d-reactive T cells, NK T cells, invariantT cells, or JαQ⁺ T cells) in the mammal relative to the correspondinglevel of T cell activity in the mammal before administration of theanti-CD1 antibody, or relative to the corresponding level of T cellactivity in an untreated mammal. In other embodiments, the methoddecreases the amount of IL-12, TNF, and/or IFN-γ and/or increases theamount of TGF-β and/or IL-10. Desirably, the decrease in the amount ofone or more of these cytokines is at least 10, 20, 30, 40, 50, 60, 80,90, or 95%. In some embodiments, the method increases the amount ofTGF-β, IL-1α, IL-10, or IL-12 by at least 10, 20, 30, 40, 50, 60, 80,90, 100, 200, 500, or even 1,000%. In particular embodiments, the methodincreases the amount of IL-12 produced by dendritic cells by at least10, 20, 30, 40, 50, 60, 80, 90, 100, 200, 500, or even 1,000%.

In another desirable embodiment, a cell type or other agent such as ananti-microbial, chemotherapeutic, immune suppressive, orimmunomodulatory compound which works in concert with the antibodies isalso administered to the mammal. In various embodiments, one or morecytokines (e.g., IL-2, IL-4, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18,IFN-α/β, IFN-γ, or GM-CSF) are also administered to the mammal.Desirably, the cytokine is administered to the mammal before, during, orafter the anti-CD1 antibody is administered to the mammal. In desirableembodiments, the cytokine is administered intramuscularly,intravenously, intraarticularly, intralesionally, subcutaneously, or byany other route sufficient to provide a dose adequate to modulate theactivity of an APC or a T cell (e.g., a CD1-reactive T cell). Desirably,the cytokine alters the ratio of Th1/Th2/immune deviation response bythe contacted T cells. In some embodiments of methods of treating orpreventing an undesired immune responses (e.g., autoimmunity, transplantrejection, graft-versus-host disease, allergy, asthma, fetal rejection,or immune complex diseases) an anti-CD1 antibody is administered incombination with an agent (e.g., TGF-β or IL-4) which alone or incombination promotes an anti-inflammatory response through an APC. Insome embodiments, an antigen such as a lipid orglycosyl-phosphatidylinositol antigen from an infectious pathogen, anantigen from a cancerous cell, a self-lipid, α-galactosylceramide, or anantigen other than α-galactosylceramide is administered to the mammal.In various embodiments, an anti-CD1 antibody and an antigen such as alipid or glycosyl-phosphatidylinositol antigen from an infectiouspathogen, an antigen from a cancerous cell, a self-lipid,α-galactosylceramide, or an antigen other than α-galactosylceramide iscontacted with the APC ex vivo, and the cell is later introduced into amammal. In other embodiments, an anti-CD40 antibody is also administeredto the mammal.

In desirable embodiments, the anti-CD1 antibody binds and activates APCsexpressing CD1d. In some embodiments, the anti-CD1 antibody onlysubstantially activates APCs expressing one CD1 molecule (e.g., onlyactivating APCs expressing one CD1 molecule selected from the groupconsisting of CD1a, CD1b, CD1c, and CD1d). For example, the antibody mayactivate APCs expressing CD1d by at least 2, 5, or 10 fold more thanAPCs expressing CD1a, CD1b, or CD1c. Desirable APCs includeCD1-expressing monocytes, macrophages, various dendritic cells, and Bcells. Other APCs include Langerhan cells, epithelial cells, andmesenchymal cells.

In a further embodiment of any of the above aspects, administering ananti-CD1 antibody includes contacting an in-dwelling device with theantibody prior to, concurrent with, or following the administration ofthe in-dwelling device to a patient. In-dwelling devices include, butare not limited to, surgical implants, prosthetic devices, andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, and continuous ambulatory peritoneal dialysis (CAPD)catheters.

In desirable embodiments, the viral infection relevant to the methods ofthe invention is an infection by one or more of the following viruses:diabetogenic encephalomyocarditis virus, Hepatitis, picornarirus, polio,HIV, coxsacchie, herpes simplex, St. Louis encephalitis, Epstein-Barr,myxovirus, JC, coxsakievirus B, togavirus, measles, paramyxovirus,echovirus, bunyavirus, cytomegalovirus, varicella-zoster, mumps, equineencephalitis, lymphocytic choriomeningitis, rabies, simian virus 40,human polyoma virus, parvovirus, papilloma virus, primate adenovirus,and/or BK.

In the desirable embodiments, the bacterial infection is due to one ormore of the following bacteria: Chlamydophilapneumoniae, C. psittaci, C.abortus, Chlamydia trachomatis, Simkania negevensis, Parachlamydiaacanthainoebae, Pseudomonas aeruginosa, P. alcaligenes, P. chlororaphis,P. fluorescens, P. luteola, P. mendocina, P. monteilii, P.oryzihabitans, P. pertocinogena, P. pseudalcaligenes, P. putida, P.stutzeri, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli,Citrobacter freundii, Salmonella typhimurium, S. typhi, S. paratyphi, S.enteritidis, Shigella dysenteriae, S. flexneri, S. sonnei, Enterobactercloacae, E. aerogenes, Klebsiella pneumoniae, K oxytoca, Serratiamarcescens, Francisella tularensis, Morganella morganii, Proteusmirabilis, Proteus vulgaris, Providencia alcalifaciens, P. rettgeri, P.stuartii, Acinetobacter calcoaceticus, A. haemolyticus, Yersiniaenterocolitica, Y pestis, Y. pseudotuberculosis, Y intermedia,Bordetella pertussis, B. parapertussis, B. bronchiseptica, Haemophilusinfluenzae, H. parainfluenzae, H. haemolyticus, H. parahaemolyticus, H.ducreyi, Pasteurella multocida, P. haemolytica, Branhamella catarrhalis,Helicobacter pylori, Campylobacterfetus, C. jejuni, C. coli, Borreliaburgdorferi, V. cholerae, V parahaemolyticus, Legionella pneumophila,Listeria monocytogenes, Neisseria gonorrhea, N. meningitidis, Kingelladentrificans, K kingae, K. oralis, Moraxella catarrhalis, M atlantae, Mlacunata, M. nonliquefaciens, M osloensis, M. phenylpyruvica,Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis,Bacteroides 3452A homology group, Bacteroides vulgatus, B. ovalus, B.thetaiotaomicron, B. uniform is, B. eggerthii, B. splanchnicus,Clostridium difficile, Mycobacterium tuberculosis, M. avium, M.intracellulare, M leprae, C. diphtheriae, C ulcerans, C. accolens, C.afermentans, C. amycolatum, C. argentorense, C. auris, C. bovis, C.confusum, C coyleae, C. durum, C. falsenii, C. glucuronolyticum, C.itnitans, C. jeikeium, C. kutscheri, C. kroppenstedtii, C. lipophilum,C. macginleyi, C. matruchoti, C. mucifaciens, C. pilosum, C. propinquum,C. renale, C riegelii, C. sanguinis, C. singulare, C. striatum, C.sundsvallense, C. thomssenii, C urealyticum, C. xerosis, Streptococcuspneumoniae, S. agalactiae, S. pyogenes, Enterococcus avium, E.casseliflavus, E. cecorum, E. dispar, E. durans, E. faecalis, E.faecium, E. flavescens, E. gallinarum, E. hirae, E. malodoratus, E.mundtii, E. pseudoavium, E. raffinosus, E. solitarius, Staphylococcusaureus, S. epiderinidis, S. saprophyticus, S. internedius, S. hyicus, S.haemolyticus, S. hominis, and/or S. saccharolyticus. In someembodiments, the bacterial infection is an infection by a bacteria otherthan listeria. Desirably, an anti-CD1 antibody is administered in anamount sufficient to prevent, stabilize, or inhibit the growth of apathogen or to kill the pathogen.

In other embodiments, the autoimmune disease is type 1 diabetes. In someembodiments, the autoimmune disease is a disease other than lupus. Inyet other embodiments, the disease treated or prevented using themethods of the invention is a condition (e.g., an autoimmune diseasesuch as diabetes or multiple sclerosis) in which downregulation of IL-12production (e.g., a reduction of at least 25, 50, 75, or 90%) isbeneficial.

Desirably, the mammal is a non-rodent mammal or a human. Other desirableanimals include mammals of laboratory or veterinary interest such asmice, rats, rabbits, pigs, goats, cattle, sheep, and horses.

It should be understood that each of the aspects of the invention applyequally to the antibodies, bifunctional antibodies, fragments ofantibodies, and derivatives of antibodies of the invention. In variousembodiments, the antibodies in an antibody combination of the inventionare simultaneously or sequentially administered to an mammal for thetreatment stabilization, or prevention of a disease or condition.

It is also contemplated that other ligands for CD1 such as polymers ofceramide or other multimeric antigens such as multimeric lipids thatcross-link CD1 on APCs can be used any of the methods or pharmaceuticalcompositions of the invention instead of, or in addition to, an anti-CD1antibody.

By “anti-CD1 antibody” is meant an antibody which recognizes and bindsCD1 (e.g., CD1a, CD1b, CD1c, and/or CD1d), but which does notsubstantially recognize and bind other molecules in a sample, e.g., abiological sample, which naturally includes other protein or cells. Thesignal in a standard ELISA assay for the binding of the antibody to CD1expressed on a cell is desirably at least 2, 3, 4, 5, 10, 15, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 200, or 500 times greater than thatfor the binding to a control cell that is not a APC cell or to a controlcell that does not express CD1. Humanized or other species forms of theantibody may be generated using standard techniques. The antibody may bepolyclonal or monoclonal.

Desirably, the antibody is “purified,” meaning it has been separatedfrom other components that naturally accompany it. Typically, theantibody is substantially pure when it is at least 50%, by weight, freefrom proteins, antibodies, and naturally-occurring organic moleculeswith which it is naturally associated. Desirably, the antibody is atleast 75%, more desirably, at least 90%, and most desirably, at least99%, by weight, pure. A substantially pure anti-CD1 antibody may beobtained, for example, by using a method of the present invention toimmunize a mammal for the generation of the antibody, by construction ofhybridoma secreting the antibody, by chemically synthesizing theantibody, or by separation of the antibody from natural sources. Puritycan be assayed by any appropriate method, as described below for theisolation of antibodies.

By “CD1” is meant a CD1 molecule such as CD1a, CD1b, CD1c, or CD1d.Desirably, the CD1 molecule has an amino acid sequence that is at least60, 70, 80, 90, 95, or 100% identical to human CD1a, CD1b, CD1c, or CD1d(Balk et al., Proc Natl Acad Sci USA 86(1):252-6, 1989; Martin et al.,Proc Natl Acad Sci USA 84(24):9189-93, 1987).

By “fragment” is meant a polypeptide having a region of consecutiveamino acids that is identical to the corresponding region of an antibodyof the invention. The fragment has the ability to bind, activate, and/orexpand APCs ex vivo or in vivo, as determined using the assays describedherein. Desirably, the number, activity, or purity of the expanded cellsis at least 20, 40, 60, 80, or 90% of that produced by a full-lengthantibody, as measured using the assays provided herein. Desirably, thebinding of the fragment to CD1 or to an APC is at least 20, 40, 60, 80,or 90% of that of a full-length antibody.

By “derivative” is meant an antibody or fragment that is modifiedchemically or through gene fusion technology or chemical synthesis sothat it is covalently linked to a toxin, therapeutically activecompound, enzyme, cytokine, radiolabel, fluorescent label, or affinitytag. The covalently linked group can be attached to the amino terminus,carboxy terminus, between the amino and carboxy termini, or to a sidechain of an amino acid in the antibody or fragment. By “affinity tag” ismeant a peptide, protein, or compound that binds another peptide,protein, or compound. In a desirable embodiment, the affinity tag isused for purification or immobilization of the derivative. In anotherdesirable embodiment, the affinity tag or toxin is used to target theantibody or fragment to a specific cell, tissue, or organ system invivo. In still another desirable embodiment, the fluorescent orradiolabel is used for imaging of the derivative. In yet anotherdesirable embodiment, the therapeutically active compound or radiolabelis used for the treatment or prevention of a disease or disorder. Inanother embodiment, the derivative or fragment of an antibody of theinvention has increased stability or increased solubility compared tothe antibody. It is also contemplated that the antibody, fragment, orderivative of the invention may be bound non-covalently to anotherantibody covalently linked to a toxin, therapeutically active compound,enzyme, cytokine, radiolabel, fluorescent label, magnetic label, oraffinity tag.

By “humanized” is meant alteration of the amino acid sequence of anantibody so that fewer antibodies and/or immune responses are elicitedagainst the humanized antibody when it is administered to a human. Forexample, the constant region of the antibody may be replaced with theconstant region of a human antibody. For the use of the antibody in amammal other than a human, an antibody of the invention may be convertedto that species format.

By “bifunctional antibody” is meant an antibody that includes anantibody or a fragment of an antibody covalently linked to anotherantibody or another fragment of an antibody. Desirably, both antibodiesor fragments bind to different CD1 epitopes or different proteinsexpressed on the same APC. Desirably, the antibody binds CD1 a, CD 1b,CD1c, CD19, CD20, CD22, CD23, CD38, CD40, CD44, CD62L, CD69, CD83, MHCmolecules, an antigen receptor, a Fc receptor, or a cytokine receptorcomponent.

By “invariant T cell” is meant a T cell having a CD1d-reactive invariantT cell antigen receptor. By “human CD1d-reactive invariant T cellantigen receptor” is meant a T cell antigen receptor that recognizesCD1d and has an alpha chain that is generated from a rearrangementbetween Vα24 and JαQ that produces little or no N-region diversity (Kentet al., Human Immunology 60:1080-1089, 1999). In mice, the invariantTCR-a chain is generated from a rearrangement between Vα14 and Jα281that produces little or no N-region diversity. The equivalentrearrangement may occur in other mammals (e.g., rats) and in birds.Although human invariant TCR-a chain pairs preferentially with Vβ11, itcan pair with other Vβs. The human CD1d-reactive invariant T cellantigen receptor recognizes CD1d, but not the closely related CD1a,CD1b, or CD1c family members (Exley et al., J. Exp. Med. 186(1):109-120,1997).

By “treating, stabilizing, or preventing a disease or disorder” ismeant, preventing an initial or subsequent occurrence of a disease ordisorder, increasing the disease-free survival time between thedisappearance of a disease or disorder and its reoccurrence, reducing anadverse symptom associated with a disease or disorder, or inhibiting orstabilizing the progression of a disease or disorder. Desirably, atleast 20, 40, 60, 80, 90, or 95% of the treated subjects have a completeremission in which all evidence of the disease disappears. In anotherembodiment, the length of time a patient survives after being diagnosedwith a disease and treated with an anti-CD1 antibody is at least 20, 40,60, 80, 100, 200, or even 500% greater than (i) the average amount oftime an untreated patient survives or (ii) the average amount of time apatient treated with another therapy survives.

By “treating, stabilizing, or preventing cancer” is meant causing areduction in the size of a tumor, slowing or preventing an increase inthe size of a tumor, increasing the disease-free survival time betweenthe disappearance of a tumor and its reappearance, preventing an initialor subsequent occurrence of a tumor, or reducing an adverse symptomassociated with a tumor. In various embodiments, the percent ofcancerous cells surviving the treatment is at least 20, 40, 60, 80, or100% lower than the initial number of cancerous cells, as measured usingany standard assay. Desirably, the decrease in the number of cancerouscells induced by administration of a therapy of the invention is atleast 2, 5, 10, 20, or 50-fold greater than the decrease in the numberof non-cancerous cells. In yet another embodiment, the number ofcancerous cells present after administration of an anti-CD1 antibody isat least 2, 5, 10, 20, or 50-fold lower than the number of cancerouscells present after administration of a vehicle control. Desirably, themethods of the present invention result in a decrease of 20, 40, 60, 80,or 100% in the size of a tumor as determined using standard methods.Desirably, at least 20, 40, 60, 80, 90, or 95% of the treated subjectshave a complete remission in which all evidence of the cancerdisappears. Desirably, the cancer does not reappear or reappears afterat least 5, 10, 15, or 20 years. In another embodiment, the length oftime a patient survives after being diagnosed with cancer and treatedwith a therapy of the invention is at least 20, 40, 60, 80, 100, 200, oreven 500% greater than (i) the average amount of time an untreatedpatient survives or (ii) the average amount of time a patient treatedwith another therapy survives.

By “bacterial infection” is meant the invasion of a host mammal bypathogenic bacteria. For example, the infection may include theexcessive growth of bacteria that are normally present in or on the bodyof a mammal or growth of bacteria that are not normally present in or onthe mammal. More generally, a bacterial infection can be any situationin which the presence of a bacterial population(s) is damaging to a hostmammal. Thus, a mammal is “suffering” from a bacterial infection when anexcessive amount of a bacterial population is present in or on themammal's body, or when the presence of a bacterial population(s) isdamaging the cells or other tissue of the mammal. In one embodiment, thenumber of a particular genus or species of bacteria is at least 2, 4, 6,or 8 times the number normally found in the mammal. The bacterialinfection may be due to gram positive and/or gram negative bacteria.

By “viral infection” is meant the invasion of a host mammal by a virus.A viral infection can be any situation in which the presence of a viralpopulation(s) is damaging to a host mammal. Thus, a mammal is“suffering” from a viral infection when an excessive amount of a viralpopulation is present in or on the mammal's body, or when the presenceof a viral population(s) is damaging to the cells or other tissue of themammal.

By “APC pathogenesis” is meant a disease or disorder that is caused orexacerbated by an activity of an APC, such as its antibody, cytokine, orgrowth factor production. APC pathogenesis may include, for example, anautoimmune pathology, such as a response to a hepatitis infection.

By “autoimmune disease” is meant a disease in which an immune systemresponse is generated against self epitopes. Some examples of autoimmunediseases include insulin dependent diabetes mellitus, rheumatoidarthritis, pemphigus vulgaris, multiple sclerosis, and myastheniagravis.

By “increasing the number, differentiation, or activity of APCs” ismeant stimulating the activity, differentiation, or expansion of thesecells by administering an anti-CD1 antibody. Desirably, the number ofAPCs belonging to the subpopulation that are present after thisadministration is at least 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 70, 90,150, or 500 fold greater than the number of these cells present afteradministration of a control antibody. In other desirable embodiments,the amount of antibody, cytokines, or growth factors produced by APCsincreases by at least 25, 50, 75, 100, 200, 500, or even 1000%. In otherembodiments, the percentage of B cells that secrete antibody increasesby at least 25, 50, 75, 100, 200, 500, or 1000%. In still otherdesirable embodiments, the proliferation of APCs increases by at least25, 50, 75, 100, 200, 500, or 1000%, as measured using a standard assay(e.g., an assay for ³H-thymidine incorporation) to measure the rate ofDNA synthesis. Desirably, the number of monocytes that havedifferentiated into macrophages increases by at least 25, 50, 75, 100,200, 500, or even 1000%. Activated macrophages can be assayed usingstandard methods, such as those that measure the ability of macrophagesto kill microorganisms or tumor cells. Exemplary standard assays formeasuring APC activity, differentiation, and proliferation can be foundin, for example, Abbas et al. (Cellular and Molecular Immunology, 2^(nd)ed., W.B. Saunders Company, Philadelphia, 1994; Current Protocols inImmunology, Wiley, 1996 and later versions).

By “preferentially modulating the expansion or activation of at leastone CD-1 expressing APC” is meant inducing or inhibiting the expansionor activation of a CD-1 expressing APC such as a monocyte, macrophage,dendritic cell, and/or B cell. The induction of the expansion of thesecell subpopulations may be measured using standard methods, as describedabove. The inhibition of the expansion of these cell subpopulations maybe determined by comparing the number of cells belonging to thesubpopulation after incubation with an anti-CD1 antibody compared to acontrol incubation without the antibody. Desirably, the number of cellsbelonging to the subpopulation present after incubation with theantibody is 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or 100 fold less thanthe number of these cells present after the corresponding controlincubation. This inhibition of cell expansion may be useful in theprevention or treatment of APC pathogenesis. The induction or inhibitionof the activation of a cell subpopulation may be assayed using standardprocedures to measure the antibody, cytokine, or growth factorproduction or cytotoxicity of the cell subpopulation. Desirably, theincrease or decrease in the antibody, cytokine, or growth factorproduction or cytotoxicity is at least 5, 10, 20, 20, 40, 50, 70, 90, or100% of the activity of the control cell subpopulation incubated in theabsence of the antibody. Desirably, the change in the size or activityof at least one cell subpopulation selected from the group consisting ofmonocytes, macrophages, dendritic cells, and B cells is least 2, 3, 4,5, 10, 15, 20, 30, 40, 50, 70, 90, 150, or 500 times greater that thecorresponding change in cells other than monocytes, macrophages,dendritic cells, or B cells or cells other than CD1d-expressing cells.

The present invention provides numerous advantages. For example, the useof an anti-CD1 antibody for the modulation of APCs eliminates therequirement for directly modulating CD1-reactive T cells, removing themas potential sources of variability and greatly simplifying theprocedure for clinical trials. The present methods also have theadvantage of requiring only a relatively small dose of anti-CD1 antibodyto be administered to activate CD1-expressing APCs rather than requiringa relatively large dose of antibody to prevent most or all T-cells frominteracting with CD1-expressing APCs.

Other features and advantages of the invention will be apparent from thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the increased frequency of disease in CD1knockout mice and protection against viral infection using an anti-CD1monoclonal antibody. Mice were infected with EMCV-D and followed asdescribed (Exley et al., Journal of Leukocyte Biology 69:713-718, 2001).Fifty mg of the anti-CD1 monoclonal antibody 3C11 or an isotype controlmonoclonal antibody was given one day prior to infection. A standardglucose tolerance test was used to determine the percentage of mice withdiabetes. Mice with glucose levels greater than the level represented bythe vertical line in the graph (˜300 g/dl; which is the mean level ofglucose for healthy mice plus 3 standard deviations were characterizedas diabetic. The administration of the anti-CD1 antibody significantlydecreased the incidence of diabetes.

FIG. 2 is a graph showing protection against virus infection with ananti-CD1 monoclonal antibody. In an independent experiment from thatdescribed in FIG. 1, mice were infected with EMCV-D and followed asdescribed (Exley et al., Journal of Leukocyte Biology 69:713-718, 2001).Fifty mg of 3C11 anti-CD1 monoclonal antibody, 3C11 and 1B1 (fromPharmEngine) anti-CD1 monoclonal antibodies, or isotype controlmonoclonal antibodies were given one day prior to infection. Mice withglucose levels greater than the level represented by the vertical linein the graph were characterized as diabetic. The administration of oneor both anti-CD1 antibodies significantly decreased the incidence ofdiabetes.

FIG. 3 is a graph showing protection against viral infection with ananti-CD1 monoclonal antibody. In the same experiment as shown in FIG. 2,mice were infected with EMCV-D and followed as described (Exley et al.,Journal of Leukocyte Biology 69:713-718, 2001). Fifty mg of 3C11anti-CD1 monoclonal antibody, 3C11 and 1B1 (from PharmEngine) anti-CD1monoclonal antibodies, or isotype control monoclonal antibodies weregiven one day prior to infection. A paralysis score of one represents noparalysis. Mice with paralysis scores greater than the level representedby the vertical line in the figure were characterized as having viralinfection-induced paralysis. The anti-CD1 antibodies significantlyreduced paralysis in wild-type mice.

FIG. 4 is a graph showing the amount of IL-12 p70 released from isolatedmouse spleen cells in the presence of IFNγ (150 pg/ml) (n=5). Mouseanti-CD1 antibodies (1B1, 19G11 and 3C11) were used to determine whethermouse CD1d positive cells release IL-12 p70 upon activation of CD1d.These results indicate that 1B1 and 19G11 antibodies activate CD1dpositive cells to release IL-12 p70 in the presence of IFNγ. In theabsence of IFNγ, there were no detectable IL-12 p70 in the supernatants.

FIG. 5 is a graph showing the amount of IL-12 p40 released from adheredPBMC in the presence of IL-4 after 3 days (n=5). The experiment wasstopped on the third day, and the supernatants were collected for IL-12p40 measurements. IL-4 has been shown to enhance CD40 induced IL-12 p40release from antigen presenting cells (APC). Adhered PBMC stimulated on51.1 bounded plates released IL-12 p40 in the presence of IL-4 (10ng/ml). This figure shows that IL-4 may enhance the release of IL-12 p40from CD1d stimulated PBMC.

FIG. 6 is a graph showing the amount of IL-12 p40 released from adheredPBMC in the presence of IFNγ (20 ng/ml) after 3 days (n=5). Theexperiment was stopped on the third day, and the supernatants werecollected for IL-12 p40 measurements. IFNγ has been shown to enhanceCD40 induce IL-12 p40 release from APC. The increase in the amount ofreleased of IL-12 p40 is much greater in the presence of IFNγ than inthe presence of IL-4. Adhered PBMC stimulated on 51.1 bounded platesreleased IL-12 p40 in the presence of IFNγ (20 ng/ml). This figure showsthat IFNγ may also enhance the release of IL-12 p40 from CD1d stimulatedPBMC.

FIG. 7 is a graph showing the amount of IL-12 p40 released from adheredPBMC after three days (n=5). The experiment was stopped on the thirdday, and the supernatants were collected for IL-12 p40 measurements.Adhered PBMC stimulated on 51.1 bounded plates released IL-12 p40. Therelease of IL-12 p40 was less than the release of IL-12 p40 in theexperiments carried out in the presence of IFNγ but greater than therelease of IL-12 p40 in the presence of IL-4. This figure alsodemonstrates that IFNγ enhances the release of IL-12 p40 from adheredPBMC that is induced by an anti-CD1d antibody.

FIG. 8 is a graph showing the amount of IL-12 P40 released from adheredPBMC in the presence of IL-4 (10 ng/ml) and IFNγ (20 ng/ml) after threedays (n=5). The experiment was stopped on the third day, and thesupernatants were collected for IL-12 p40 measurements. Adhered PBMCstimulated on 51.1 bounded plates released IL-12 p40. The release ofIL-12 p40 was less than the release of IL-12 p40 in the experimentscarried out in the presence of IFNγ. This figure suggests that IFNγ mayenhance the anti-CD1d antibody induced release of IL-12 p40 more than acombination of IFNγ and IL-4.

FIG. 9 is a graph showing the amount of IL-12 p40 released from adheredPBMC, in the presence of IL-4 (10 ng/ml) after 8 days (n=5). Theexperiment was stopped on the eighth day, and the supernatants werecollected for IL-12 p40 measurements. Adhered PBMC stimulated on 51.1bounded plates released IL-12 p40 in the presence of IL-4 (10 ng/ml).This figure shows that IL-4 is may enhance the release of IL-12 p40 fromCD1d stimulated PBMC.

FIG. 10 is a graph showing the amount of IL-12 p40 released from adheredPBMC in the presence of IFNγ (20 ng/ml) after eight days (n=5). Theexperiment was stopped on the eighth day, and the supernatants werecollected for IL-12 p40 measurements. IFNγ has been shown to enhanceCD1d induce IL-12 p40 released from APC as shown above. Adhered PBMCstimulated on 51.1 bounded plates released IL-12 p40 in the presence ofIFNγ (20 ng/ml). The increase of IL-12 p40 was much greater in thepresence of IFNγ than in the presence of IL-4. This figure shows thatIFNγ may enhance the release of IL-12 p40 release from CD1d stimulatedPBMC. The amount of released IL-12 p40 was similar to the amountreleased at an earlier time point (3 day).

FIG. 11 is a graph showing the amount of IL-12 p40 released from adheredPBMC after eight days (n=5). The experiment was stopped on the eighthday, and the supernatants were collected for IL-12 p40 measurements.Adhered PBMC stimulated on 51.1 bounded plates released IL-12 p40. Therelease of IL-12 p40 was less than the amount released in the presenceof IFNγ (FIG. 10) but greater than the amount released in the presenceof IL-4 (FIG. 9). This figure shows also that IFNγ enhances the releaseof IL-12 p40 release from CD1d stimulated PBMC.

FIG. 12 is a graph showing the amount of IL-12 p40 released from adheredPBMC in the presence of IL-4 (10 ng/ml) and IFNγ (20 ng/ml) after eightdays (n=5). The combination of cytokines IL-4 and IFNγ did not inducethe release of IL-12 p40. Later experiments were all carried out usingthe early time points and with IFNγ since this condition results in alarger measurable production of IL-12 p40 from PBMC.

FIG. 13 is a graph showing the amount of IL-12 p70 released from C1Rd inthe presence of PMA (2.5 ng/ml) after two days (n=5). C1Rd cells wereunable to release IL-12 p70 upon CD40 or CD1d stimulation under theconditions tested.

FIG. 14 is a graph showing the amount of IL-12 p40 released from THP-1cell with and without PMA (0.1 ng/ml), 2% serum media, and IFNγ (20ng/ml) after two days (n=5). There were no significant differences inthe experiments carried out with or without PMA.

FIG. 15 is a graph showing the amount of IL-12 p40 released from THP-1cell with PMA/PDBu (1 ng/ml), 2% serum media, and IFNγ (20 ng/ml) aftertwo days (n=5). There were no significant differences in the experimentscarried out with or without PMA/PDBu.

FIG. 16 is a graph showing the amount of IL-12 p40 released from THP-1induced by different antibodies in the presence of IFNγ (20 ng/ml) onday 1 (n=5). A variety of anti-CD1 antibodies (VIT6, 4A7.6, F10/21A3 and51.1) and an anti-CD40 antibody were used. After one day, the onlyantibody that was able to stimulate the cells to produce IL-12 p40 was51.1. The amount of IL-12 p40 released was comparable to the amountreleased from cells stimulated with LPS.

FIG. 17 is a graph showing the amount of IL-12 p40 released from THP-1induced by different antibodies in the presence of IFNγ (20 ng/ml) afterthree days (n=5). A variety of anti-CD1 antibodies (VIT6, 4A7.6,F10/21A3 and 51.1) and an anti-CD40 antibody were used. After 2 moredays the only antibody that was able to stimulate the cells to produceIL-12 p40 was 51.1. The amount of IL-12 p40 seen was comparable to cellswhich were stimulated with LPS.

FIG. 18 is a graph showing the amount of IL-12 p40 released from THP-1using all soluble antibodies and serum free media without IFNγ after twodays (n=5). Cross-linking of soluble antibodies using anti-mouse IgG didnot induce IL-12 p40 release from THP-1 cells but inhibited the releaseof IL-12 p40 from all cells tested, including the positive control(LPS). These results suggest that an anti-CD1 antibody bound to a platemay stimulate IL-12 production more than the corresponding solubleanti-CD1 antibody.

FIG. 19 is a graph demonstrating that the induction of IL-12 p40 releaseby the 51.1 antibody was not due to endotoxins (n=5). The 51.1 antibodywas heated at 100° C. for 30 minutes to destroy the antibody structureto determine whether cell activation was solely due to the antibody andnot due to endotoxins. After two days, the amount of released IL-12 p40was measured. The results demonstrate that the boiled antibody wasunable to stimulate the cells, and thus, the release of IL-12 p40 wassolely due to CD1d activation.

FIG. 20 is a graph demonstrating the effect of an NFκB inhibitor (PDTC)on the release of IL-12 p40 induced by the 51.1 anti-CD1 antibody andLPS (n=5). This figure shows that NFκB is involved in the transcriptionof IL-12 p40.

FIG. 21 is a graph showing the amount of IL-12 p40 released from THP-1cells in the presence of IFNγ (10 ng/ml) and 2% serum media after twodays (n=5).

FIG. 22 is a graph showing the amount of IL-12 p40 released from THP-1cells IFNγ (10 ng/ml) and 2% serum media (1 ng/ml PMA) after two days(n=5).

FIG. 23 is a picture of a Western blot showing the present of CD1d inTHP-1 cells. The CD1d band shown on the blot indicates that THP-1 cellshave CD1d either in the cells or expressed on the cell surface.

FIG. 24 is a picture of a Western blot showing the increased levels ofP—IκB in THP-1 cells during stimulation with the 51.1 anti-CD1dantibody. No increase of P—IκB was observed on the western blot when thecells were put on plates that were not coated with protein G prior to51.1. In contrast, plates that were coated with protein G prior to 51.1were able to stimulate the cells. The level of p-IκB in cells after 45minutes of 51.1 stimulation was similar to the levels found in LPSstimulated cells. These results indicate that cross-linking of CD1d withprotein G is required for NFκB activation.

FIG. 25 is a bar graph showing the relatively low amount of IL-1αreleased from adhered PBMC in the presence of IFNγ (20 ng/ml) (n=4). Theexperiment was stopped after one day of incubation, and the supernatantwas collected for IL-1α measurements. The 51.1 anti-CD1d antibodystimulated the release of IL-1α in an amount similar to that releasedfrom LPS-stimulated cells.

FIGS. 26A-26D are graphs of a FacScan showing the presence of externalCD1d on the cell surface of THP-1 cells.

FIGS. 27A-27D are graphs of a FacScan showing the absence of externalCD1d on the cell surface of C1 Rmocks (negative control).

FIGS. 28A-28D are graphs of a FacScan showing the presence of externalCD1d on the cell surface of C1Rd cells (positive control).

FIG. 29 is a bar graph showing the amount of IL-12 p40 released fromTHP-1 cells in the presence of IFNγ (20 ng/ml) (n-4). The experiment wasstopped after one day of incubation, and the supernatant was collectedfor IL-12 p40 measurements. The 51.1 anti-CD1 antibody on protein Gcoated plates was able to cross-link CD1d on THP-1 cells. Cross-linkingof CD1d stimulates THP-1 cells to produce IL-12 p40. Cells from controlplates that were not coated with protein G prior to 51.1 anti-CD1antibody binding were unable to stimulate THP-1 cells. The data showthat cross-linking of CD1d is important for induction of IL-12 p40release by the 51.1 anti-CD1 antibody.

FIG. 30 is a bar graph demonstrating the effect of an NFκB inhibitor(parthenolide, 0.1 μM) on the release of IL-12 p40 by THP-1 cells thatis induced by the 51.1 anti-CD1 antibody or LPS (n=4). These dataindicate that NFκB is involved in the transcription of IL-12 p40.

FIG. 31 is a bar graph showing the relatively low amount of IL-10released from adhered PBMC in the presence of IFNγ (20 ng/ml) (n=4). Theexperiment was stopped after one day of incubation, and the supernatantwas collected for IL-10 measurements. The 51.1 anti-CD1 antibodystimulated the release of IL-10. In particular, the amount of IL-10released after stimulation with the 51.1 anti-CD1 antibody was similarto the amount of IL-10 released after stimulation with LPS.

DETAILED DESCRIPTION

We have made the surprising discovery that an anti-CD1 antibody can beused to directly activate antigen presenting cells (APCs; e.g.,CD1-expressing monocytes, macrophages, dendritic cells, and B cells)without the need to directly modulate the activity of CD1-reactive Tcells. For example, administration of an anti-CD1 antibody resulted inthe production of therapeutic cytokines and protective responses in aviral model. In particular, anti-CD1 antibodies induced IL-12, IL-1α,and IL-10 and protected mice against lethal challenge in an acute viralinfection model, the picornavirus EMCV. In vitro results alsodemonstrated that anti-CD1 antibodies are more potent than CD40 ligationin stimulation of monocytes for production of bioactive IL-12. CD1antibodies can also synergize with antibodies such as anti-CD40antibodies to stimulate monocytes in vitro. Even in the absence ofanti-CD40 antibodies, anti-CD1 antibodies can activate dendritic cellsto produce bioactive IL-12.

The generality of CD1 activation was confirmed using panels of CD1antibodies. Several monoclonal antibodies to different CD1 epitopesactivated the target cells. IFN-γ enhances sensitivity so that ananti-CD1 antibody alone activates APCs without the need for CD40ligation.

Thus, anti-CD1 antibodies represent novel therapeutic approaches tooptimizing protective responses without the need to directly modulatethe activity of CD1-reactive T cells (e.g., without having tosignificantly or completely inhibit the binding of CD1-reactive T cellsto CD1-expressing APCs). The present invention uses ligands for CD1(e.g., antibodies) to directly bind and activate monocytes and otherAPCs, leading to production of therapeutic cytokines and by cell-contactto stimulation of protective responses. Anti-CD1 antibodies can be usedfor in vivo or alternately in vitro activation of APC followed byre-infusion to induce protective responses against viral, bacterial,yeast, and parasitic infectious diseases and cancer and to positivelyinfluence undesirable responses such as autoimmunity and allergicresponses. The administration of anti-CD1 antibodies of the inventionmay be performed alone or in conjunction with the administration ofother therapeutics, such as cytokines, tumor vaccines, or otherantibodies reactive with APCs.

Anti-CD1 antibodies may be administered to a mammal, such as a human,for the prevention or treatment of autoimmune diseases, infectiousdiseases, allergies, asthma, inflammatory conditions, graft versus hostdisease, graft rejection, immunodeficiency disease, spontaneousabortion, pregnancy, or cancer. Anti-CD1 antibodies may be used toactivate CD1-expressing APCs and thereby stimulate protective responsesof CD1-reactive T cells. For example, reduced levels of certain T cellssuch as invariant NK T cells are found in prostate cancer, multiplesclerosis, HIV, and type 1 diabetes patients (WO 01/98357, publishedDec. 27, 2001). Additionally, a reduced number of Vα24⁺ CD161⁺ T cellshas been previously reported for melanoma patents (Kawano et al., CancerRes. 59:5102, 1999); thus, the loss of invariant NK T cell function maybe a general finding in advanced cancer.

In addition to, or instead of, administering an anti-CD1 antibody to amammal, APCs that have been incubated in vitro with an anti-CD1 antibodycan be administered to a mammal to increase the number of active APCs inthe mammal. Desirably, the APCs are administered to the same mammal fromwhich they were isolated.

Anti-CD1 antibodies may also be administered with any commerciallyavailable vaccine to enhance the immune response to an infection. Thus,anti-CD1antibodies can be used to increase the effectiveness of avaccine for the prevention or treatment of an infection, such as abacterial or viral infection.

Alternatively, antibodies that bind and inhibit the expansion or anactivity of APCs, such as antibody or cytokine production, may be usedto inhibit APC cell pathogenesis in a mammal. The immune responseinduced by infectious agents, such as Hepatitis viruses, can causedamage which may be minimized by the inhibition of these cells. Examplesof some of the antibodies that may inhibit these cells includemonovalent or Fab molecules or antibodies that are conjugated to a toxinor radiolabel that damages the cells upon binding of the antibody to thecells.

In addition to administering an anti-CD1 antibody or ex vivo expandedAPCs, cytokines can also be administered to a mammal to further modulatethe immune system. Using a cytoline, such as IL-12, IL-15, or IL-18,which is known to bias T cells towards Th1 responses is expected toincrease the effectiveness of the present methods in the prevention ortreatment of cancer, infectious disease, allergies, asthma, pregnancy,and inflammation. Alternatively, any other cytokine, such as IL-2, IL-4,IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IFN-α/β, IFN-γ, and GM-CSF, maybe used to bias the T cells towards Th2 responses for the prevention ortreatment of autoimmune diseases and graft versus host disease for whichTh2 responses are protective. Alternatively, the cytoline may be used tobias the T cells away from Th1 and Th2 responses and towards immunedeviation responses which may contribute to the maintenance ofpregnancy. Immune deviation responses include the suppression of anongoing immune response, such as a response at a immune-privileged site.TGF-β and IL-10 are examples of cytokines that may participate in immunedeviation responses (Sonoda et al., supra).

The following examples are to illustrate the invention. They are notmeant to limit the invention in any way.

EXAMPLE 1 Generation of anti-CD1 Antibodies

The generation of the 3C11 and 51.1 anti-CD1 antibodies has beendescribed previously (see, for example, Bleicher et al., Science250:679-682, 1990; Exley, Immunology 100:37-47, 2000; Kasinrerk et al.,J Immunol 150(2):579-84, 1993; Dezutter-Dambuyant et al., Res Immunol140(4):377-90, 1989; Kahn-Perles et al., J. Immunol. 134(3):1759-65,1985; Porcelli et al., Nature 241(6241):447-50, 1989).

The 3C11 monoclonal antibody used in the experiments described hereinwas obtained from Dr. Steven Porcelli (formerly at Brigham & Women'sHospital, Mass.; now at Einstein College of Medicine, NY), who generatedthe antibody using antigen provided by Dr. Steven Balk. Additionally,the 1B1 anti-mouse CD1 monoclonal antibody is commercially availablefrom Pharmingen.

For the production of additional anti-CD1 antibodies and hybridomas, apeptide from CD1, the entire CD1 protein, or APCs can be used. Forexample, an antigenic peptide is coupled to a carrier (e.g., a keyholelimpet hemocyanin carrier) and administered to an animal (e.g., alaboratory animal or an animal of veterinary interest) in one or moredoses. Desirable routes of administration include intraperitoneally,intramuscular, intradermal, and subcutaneous. The dose and frequency ofadministration can be determined using standard procedures. Examples ofmammals that may be used for the production of antibodies include mice,rats, rabbits, pigs, goats, sheep, horses, and cattle. Examples of birdsthat may be used include chickens and turkeys. The host animals may bewild-type animals, or they may be animals that have a reduced level orthat lack CD1 or APCs. Other animals that may be used include animalsthat naturally, through genetic modification, or depletion lack CD1 orAPCs. Any other animal that is capable of producing antibodies may alsobe used for the production of antibodies. If desired, the same peptide,another CD1 peptide, full-length CD1, or APCs may be administered asbooster injections. Serum from the animal is tested for antibodies thatbind the CD1 peptide or APC using a standard ELISA assay. Hybridomas aregenerated from seropositive animal spleens using standard techniques.ELISA positive hybridoma wells are further tested by FACS to comparebinding of the antibodies to CD1 or APCs versus negative controls. Cellsproducing antibodies that bind CD1 or APCs but not the negative controlsare cloned and fused to immune spleen cells using standard techniques. Agroup of hybridomas secreting monoclonal antibodies that specificallyrecognize CD1 (e.g., CD1d) are then identified using the ELISA assay. Togenerate antibodies that enhance production of IL-12 by THP-1 cells,hybridomas from animals desirably immunized with an APC or a syntheticantigen may be screened for the ability to simulate APC responses suchas IL-12 or TGF-β production by measuring the activity or cytokinelevels in the supernatants of the hybridomas. These stable monoclonalantibody secreting hybridomas clones may be used by one skilled in theart for the production and purification of clinical trial quality andquantity of these monoclonal antibody reagents.

Anti-CD1 antibodies can be purified from antiserum, ascites fluid, orhybridomas supernatant by one skilled in the art using standardtechniques such as those described by Ausubel et al. (Current Protocolsin Molecular Biology, volume 2, p. 11.13.1-11.13.3, John Wiley & Sons,1995). The antibody is desirably at least 2, 5, or 10 times as pure asthe starting material, as measured using polyacrylamide gelelectrophoresis, column chromatography, optical density, HPLC analysis,or western analysis to detect a reduction in the amount of contaminatingproteins or ELISA to detect an increase in specific activity for bindingto CD1 or APCs.

The resulting antibodies may be used for clinical applications involvingother animals of the same genus or species as the host animal used forantibody production. Additionally, the antibodies of the invention maybe used in applications involving animals of a different genus as thehost animal. For example, antibodies that are produced in bovines may beused for the treatment or prevention of disease in other bovines or inother mammals, such as humans.

EXAMPLE 2 Prevention and Treatment of Viral Infection using anti-CD1Antibodies

The picornavirus, diabetogenic encephalomyocarditis virus (EMCV-D),causes lethal, acute disease in young male mice of appropriate strains.Female and older male mice are resistant. The severity of EMCV-D-induceddisease varies in different genetic backgrounds, BALB/c being mostsensitive and C57/B16 being most resistant. Contributions of NK cells,macrophages, and T cells to responses against various EMCV strains alsovary in different strains. EMCV-D-induced disease can be measured byhind-limb paralysis (a manifestation of encephalitis) andglucose-tolerance testing (diabetes), reflecting acute, cytopathiceffects of the virus on neuronal cells and islet cells, respectively. Afurther manifestation of EMCV-D infection is myocarditis. EMCV inducedmyocarditis involves limited, direct, viral damage in conjunction withextensive mononuclearcell infiltration.

To determine the ability of anti-CD1 antibodies to protect against viralinfection, mice were administered one or more anti-CD1 antibodies priorto infection with EMCV-D (Exley et al., Journal of Leukocyte Biology69:713-718, 2001). Briefly, BALB/c-backcrossed, CD1d-KO mice (F8), andJα281-KO mice (F 10) were administered fifty microgram of anti-CD1monoclonal antibody 3C11, anti-CD1 monoclonal antibodies 3C11 and 1B1,or isotype control antibody one day prior to viral infection (Smiley etal, Science 275:977-979, 1997; Cui et al., Science 278:1623-1626; 1997).Wild-type and knockout (1293C57/B16)F2 or BALB/c mice were infectedintraperitoneally (i.p.) with 800 plaque-forming units (PFU) EMCV-D(Giron et al., Proc. Soc. Exp. Biol. Med. 173:328-331, 1983). The micewere then examined to determine the frequency of paralysis and diabetes.Paralysis score 1 denotes no paralysis; 2 denotes limp or partial use ofone paw; 3 denotes one completely paralyzed hind paw; 4 denotes loss oftwo hind limbs; and 5 denotes paralysis of three or four paws. Glucosetolerance testing was performed by i.p. injection of 2 g/Kg glucose withblood collected into glucosidase inhibitor-treated tubes at one hour(Giron et al., supra). Organs were fixed for histology at this time.

As illustrated in FIG. 1, administration of the anti-CD1 monoclonalantibody 3C11 prevented diabetes in wild-type mice infected with EMCV-D.In contrast, 33% of the wild-type mice that were administered a controlantibody prior to EMCV-D infection became diabetic. These results wereconfirmed in an independent experiment. Additionally, the administrationof both 3C11 and 1B1 anti-CD1 monoclonal antibodies further reducedglucose levels in viral infected mice (FIG. 2). The administration of3C11 antibody or 1B1 and CD1 antibodies also reduced the severity ofparalysis induced by EMCV-D infection (FIG. 3).

FIG. 1 also demonstrates that CD1 knockout mice have a much higherincidence of diabetes (100%) compared to uninfected, wild-type mice(0%). This result supports the important role of CD1-expressing cells inthe prevention of undesired immune responses, such as viralinfection-induced diabetes.

EXAMPLE 3 Ability of Anti-CD1 Antibodies to Enhance Production ofCytokines by Murine and Human Antigen Presenting Cells

IL-12 is an inducible cytokine composed of 35 kDa and 40 kDa subunitsthat are critical in inducing helper type 1 T cells during initialimmune responses to pathogens. The 40 kDa subunit, expressed byactivated antigen presenting cells, is induced by pathogens. Control ofIL-12 p40 expression is therefore important for understanding resistanceand susceptibility to pathogens.

To demonstrate the ability of anti-CD1 antibodies to increase theproduction of IL-12, mouse CD1d positive splenocyte cells, humanperipheral blood mononuclear cells (PBMC), and human THP-1 monocyticcell line were analyzed. PBMC were obtained from healthy adultvolunteers of both sexes on Ficoll Hypaque density gradients andcultured in complete medium consisting of RPMI 1640 and 10% FBS. THP-1is a human monocytic leukaemia cell line. Compared to other humanmyeloid cell lines, such as HL-60 and U937, THP-1 cells express CD1d ontheir surfaces. Because of this characteristic, the THP-1 cell lineprovides a valuable model for studying the mechanism involved in CD1dcrosslinking by measuring the IL-12 and phospho kinases in the cell.THP-1 cells was cultured in complete medium consisting of RPMI 1640 and10% FBS.

For the activation of these target cells, an anti-CD1 antibody was boundto a plate and then incubated with the cells. In particular, 98-wellplates were coated with 10 μg/ml protein G overnight at 4° C. Wells werewashed with PBS and blocked with BSA at room temperature for four hours.Antibody (10 μg/ml) was added to each well for four hours at roomtemperature. The wells were washed again to remove the unboundantibodies, and 1.5×10⁵ to 2×10⁵ cells were added to each well in RPMI1640 culture media.

To confirm the presence of CD1d on the surface of THP-1 cells,fluorescent staining of cells were performed using the 51.1 monoclonalanti-CD1d antibody and fluorescently labelled anti-human IgG (H+ L) HAS.Stained cells were analysed on a FACScan cytometer (FIGS. 26A-26D,27A-27D, and 28A-28D). To measure the level of CD1d expressed by THP-1cells or to measure the level of P—IκB induced by an anti-CD1 antibody,equal amounts of cytosolic extracts were separated on a 10% SDS-PAGE geland transferred to a nitocellulose membrane (FIGS. 23 and 24). Themembranes were blocked in 5% BSA in TBST, incubated with the primaryantibody and detected with horseradish peroxidase-conjugated secondaryantibody and enhanced chemilluminesence substrate.

To measure the increase induction of IL-12 production by an anti-CD1antibody, the concentrations of P40 and P70 forms of IL-12 in culturedsupernatants were measured with a standard sandwich ELISA techniqueusing combinations of monoclonal antibodies to different epitopes ofeach cytokine (FIGS. 4-22).

The results from the above experiments demonstrate that the 51.1anti-CD1 antibody induced production of IL-12 (FIGS. 4-11, 14, 15, 21,and 22). FIG. 29 illustrates the results of a similar experiment inwhich the 51.1 anti-CD1 antibody bound to protein G coated platesinduced the release of IL-12 by THP-1 cells in the presence of IFNγ. TheNFκB inhibitor parthenolide caused a reduction in the amount of IL-12p40 that was released by THP-1 cells due to incubation in the presenceof the 51.1 anti-CD1 antibody or LPS (FIG. 30), indicating that NFκB isinvolved in the transcription of IL-12 p40.

The 51.1 anti-CD1 antibody was more potent than CD40 ligation in thestimulation of monocytes to produce bioactive IL-12. Anti-CD1 antibodiescan also synergize with antibodies such as anti-CD40 antibodies tostimulate monocytes in vitro. IL-4 and IFNγ also increased the abilityof anti-CD1 antibodies to enhance production of IL-12. Even in theabsence of anti-CD40 antibodies, anti-CD1 antibodies can activatedendritic cells to produce bioactive IL-12.

The generality of CD1 activation was confirmed using panels of CD1antibodies. Several monoclonal antibodies to different CD1 epitopesactivated the target cells. These results demonstrate that anti-CD1antibodies are useful in the treatment and prevention of conditions forwhich increased levels of IL-12 are desirable. Examples of suchconditions include cancers and infections by pathogens such as bacteria,viruses, or yeast.

In addition to stimulating the release of large amounts of bioactiveIL-12, the 51.1 anti-CD1 antibody can also stimulate the release of lowlevels of IL-10 and IL-1α (FIGS. 25 and 31). IL-10 has a wide range ofin vivo biological activities and is a key regulatory cytokine ofimmune-mediated inflammation. This cytokine has been shown to inhibitthe functions of key elements of both innate and acquired immuneresponses. IL-10 also enhances the function of a recombinantpoxvirus-based anti-cancer vaccine and may represent a potentialadjuvant in the vaccination against human cancers using recombinantpoxvirus-based vaccines. IL-1 has a broad range of physiological effectsand is released early in a disease or injury process. For example, IL-1is an important mediator of inflammation and tissue damage in multipleorgans. LPS is a more potent inducer of IL-1, but only stimulatescomparable or lower levels of IL-12. Therefore, the ability of the 51.1anti-CD1 antibody to selectively stimulate the production of IL-12supports its usefulness for the treatment or prevention of a variety ofdiseases.

EXAMPLE 4 Administration of Antibodies for In Vivo Activation orExpansion of APCs

One or more anti-CD1 antibodies may be administered to a mammal,possibly in addition to the administration of a cytokine, for the invivo expansion of APCs for the treatment or prevention of an autoimmunedisease, viral infection, bacterial infection, parasitic infection,infection by a eukaryotic pathogen, allergy, asthma, inflammatorycondition, graft versus host disease, graft rejection, immunodeficiencydisease, spontaneous abortion, pregnancy, or cancer. As described inExample 7, all modes of administration, dosing, and frequency arecontemplated. Examples of doses of anti-CD1 antibodies that may beadministered to a mammal such as a human include doses that fall withinone of the following ranges: 0.1 to 10 mg/kg, 0.1 to 0.4 mg/kg, 0.1 to 1mg/kg, or 1 to 4 mg/kg, inclusive.

The pharmaceutical compositions containing one or more antibodies of theinvention may be prepared as described previously in Remingtion'sPharmaceutical Sciences by E. W. Martin. Pharmaceutical stabilizingcompounds, delivery vehicles, or carrier vehicles may be used. Forexample, human serum albumin or other human or animal proteins may beused. Phospholipid vesicles or liposomal suspensions are possiblepharmaceutically acceptable carriers or delivery vehicles. Thesecompositions can be prepared according to methods known to those skilledin the art.

An antibody of the invention that is covalently linked to a fluorescentlabel or radiolabel may be used to visualize the in vivo distribution,quantity, or migration of APCs. This imaging of APCs may be used toidentify subjects who are at risk for or have an autoimmune disease,viral infection, bacterial infection, parasitic infection, infection bya eukaryotic pathogen, allergy, asthma, inflammatory condition, graftversus host disease, graft rejection, immunodeficiency disease,spontaneous abortion, pregnancy, or cancer. Alternatively, this methodmay be used to determine the effect of a therapy for one of the abovediseases on APCs.

EXAMPLE 5 Optional Cytokine Treatment to Alter the Th1/Th2/ImmuneDeviation Response Ratio of T Cells

While not meant to limit the invention to a particular theory, a workinghypothesis is that NK T cells, CD1d-reactive T cells, or JαQ⁺ T cellsproducing high levels of IL-4 possibly in conjunction with other “Th2”cytokines) will bias toward Th2 immune responses, while high IL-12(induced from APCs by activated CD1d-reactive T cells) and/or IFN-γ(produced by the T cells themselves) relative to IL-4 will drive (or atleast be a marker of invariant T cells that will drive) immune responsesagainst tumors or infectious pathogens.

In addition to modulating the immune system by administering an anti-CD1antibody, NK T cells, CD1d-reactive T cells, or JαQ⁺ T cells may bebiased towards Th1-like NK or lymphokine-activated killer (LAK)-likecytotoxicity of invariant T cells, as well as, IFN-γ production topotentially augment their in vivo anti-tumor or anti-pathogen activity.For example, IL-12 augments both IFN-γ production and, significantly,NK/LAK-like cytotoxicity of invariant T cells have recently beenpublished (van der Vliet et al., Immunology 98:557-63, 1999; WO01/98357). IL-15, IL-18, and type 1 INFs are also known to enhance Th1polarization of human T cells. Other cytokines or combinations ofcytokines that may bias NK T cells, CD1d-reactive T cells, or JαQ⁺ Tcells towards Th1, Th2, or immune deviation responses include IL-2,IL-4, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-18, IFN-α/β, IFN-γ, andGM-CSF. The Th2 response may be desirable for the prevention ortreatment of an autoimmune disease. Alternatively, for the maintenanceof pregnancy, cytokines may be used to bias NK T cells, CD1d-reactive Tcells, or JαQ⁺ T cells towards immune deviation responses instead of Th1or Th2 responses.

After administration of one or more of these cytokines, the T cells in asample from a mammal may be functionally tested for secretion of L-4,IL-10, GM-CSF, and IFN-γ. The regulation of cytotoxicity againstCD1d⁺(CIR, CD1d), NK′ targets (JY, K562, 721.221, and YAC-1), or LAKtargets by cytokine supplementation may also be determined (Exley etal., supra (1997); Exley et al., J. Exp. Med. 188:867-876, (1998)).

EXAMPLE 6 Methods for Determining the Effect of Antibodies on theTargeted APC Subpopulation In Vivo

Any of the antibodies of the invention may be tested in an in vivoanimal or primate model to determine the pharmacological andpharmacokinetic properties of the antibodies. For example, thehalf-life, bio-distribution, and efficacy of the antibody may bedetermined.

One possible method involves the administration of human APCs or anyother APC population of interest to a SCID or otherwise immune-deficientanimal such as a mouse and administration of an anti-CD1 antibody to theanimal to determine whether the antibody modulates the activity ornumber of the administered APCs in vivo.

In particular, a population of APCs that contains 1-10 million APCs ofinterest is administered i.v. or to any site of interest. One or moreantibodies are administered, prior to, concurrent with, or followingadministration of the APCs. For example, the antibody may beadministered at any point during the lifetime of the administered APCsin the host animal. Approximately 1-100 μg of the antibody isadministered in the same site or in a different site as the site ofadministration of the APCs. If detectably labeled antibodies are used,the location and amount of administered antibody and/or APCs may bemonitored in vivo based on fluorescence or radioactivity. Additionally,histology, immunological, and/or biochemical measurements may beperformed ex vivo on tissues from the animal. The biological activity ofthe antibody or APC subpopulation may be measured by analyzing theamount or activity of antibodies, growth factors, or cytokines in aserum or tissue sample. Moreover, the activation of other cells, such asT cells, by the administered APC subpopulation may be measured. Forexample, the number of CD1-reactive T cells may be measured by FACSsorting.

Other animal or primate models of immune or inflammatory disorders andpathogenic infections are known to those skilled in the art and can bereadily used to determine the ability of an anti-CD1 antibody orantibody combination or ex vivo expanded APCs (Example 7) to prevent ortreat the condition. For example, an animal model of cancer is disclosedby Karnbach et al. (J Immunol 167(5):2569-76, 2001), and an animal modelof bacterial infection is disclosed by Lehmann et al. (J Immunol167(9):5304-15, 2001).

Schwartzman Reaction

One of the classic examples of necrotizing inflammation is theSchwartzman reaction, in which lipopolysaccharide (LPS) is introducedfirst into rabbit skin and then, 24 hours later, followed by a secondintravenous dose of the same LPS (McFadden and Lucas, WO 96/33730; filedApr. 19, 1996). Within hours after the second LPS injection,infiltrating macrophages induce a reproducible necrotizing response atthe site of the primary injection which is highly reproducible andreadily quantified. The ability of an anti-CD1 antibody or antibodycombination to inhibit this inflammation may be examined.

New Zealand White female rabbits weighing 3 kg are injected withlipopolysaccharide (LPS) of the E. coli serotype 0111:B4 (Sigma) andCBP-I (T7) protein which had been purified to homogeneity using columnchromatography. Eight intradermal injections (0.1 ml each) of 50-100 μgLPS in the presence and absence of an anti-CD1 antibody is applied tothe back of the rabbit; there are 4 injection sites on each side,separated by about 2.5 cm. Twenty-four hours later, 100 μg of LPS isadministered to the rabbit intravenously in the marginal ear vein. About4-6 hours after the intravenous injection, necrotic inflammationtypically develops at the sites of intradermal injection of LPS (in theabsence of an anti-CD1 antibody). As soon as the inflammation issignificant, the rabbit is sacrificed by a lethal injection of euthanol.The size and redness of the lesions are assessed, and tissue samples arecollected.

Models of Injury Induced Atherosclerosis

Inflammation has been associated with accelerated atherosclerotic plaquedevelopment in the arterial wall. There is a high rate of plaquerecurrence, restenosis, after the use of balloon angioplasty and otherrelated angioplasty devices designed to open occluded arteries.Accelerated atherosclerotic plaque growth also has been reported underconditions leading to arterial injury, viral infections, vasculitis,homocystinuria, diabetes melitis, hypertension, hyperlipideuria,smoking, and immune complex generated disorders. Thus, anti-CD1antibodies can also be tested in the rat and rabbit models of injuryinduced atherosclerosis disclosed by McFadden and Lucas (WO 96/33730;filed Apr. 19, 1996).

EXAMPLE 7 Expansion and Re-Introduction of Ex Vivo Expanded APCs intoMammals

APCs from a peripheral blood sample (20 ml or from the product ofleukopheresis) from a mammal may be enriched prior to ex vivo expansionusing FACS sorting or immunoaffinity purification or expanded directlyusing an anti-CD1 antibody, as described in the previous examples.Additionally, IL-2, IL-7, or a mitogen may be added to stimulate cellexpansion. IL-4/GM-CSF may be added to stimulate monocytic cells. Ifnecessary, a secondary ex vivo expansion, possibly after a secondenrichment step, may be conducted under conditions used for the primaryexpansion to increase both cell number and purity. Exemplary amounts ofanti-CD1 antibody for stimulation of APCs such as human APCs includesamounts in one of the following ranges: 0.1 to 10 μg, 0.1 to 1 μg, 1 to5 μg, or 5 to 10 μg per one ml of cells with a concentration of 0.5 to1×10⁶ cells per ml. After stimulation, the cells may be assayed forpurity and for production of an antibody, cytokine, or growth factor asdescribed above. Desirably at least 10⁶, more desirably at least 10⁸,and most desirably at least 10⁹ APCs are obtained after expansion.Desirably, the APCs are at least 60%, 80, or 90% pure, based on thepresence of CD1 and/or other APC markers, and maintain the production ofcytokines, antibodies, and/or growth factors.

If secondary stimulations do not yield adequate cell numbers, thenadditional rounds of stimulation may be used. Alternatively, the numberof starting cells may be increased by using larger blood samples orthrough leukopheresis. In this case, an initial enrichment of APC may beperformed by positive selection using the 27.1, 42.1, 51.1 or similarmonoclonal antibodies conjugated to beads. To increase purity, the APCsmay also be purified by FACS or antibody conjugated to beads prior tore-infusion. Additionally, the therapeutic potential of cellularreinfusion of expanded polyclonal APC lines may be compared to that ofexpanded APC clones, or pools thereof.

It is not intended that the administration of ex vivo expanded APCs belimited to a particular mode of administration, dosage, or frequency ofdosing; the present mode contemplates all modes of administration,including intramuscular, intravenous, intraarticular, intralesional,subcutaneous, or any other route sufficient to provide a dose adequateto prevent or treat an autoimmune disease, viral infection, bacterialinfection, parasitic infection, infection by a eukaryotic pathogen,allergy, asthma, inflammatory condition, host versus graft disease,spontaneous abortion, pregnancy, or cancer. Desirably, the cells arere-introduced into the mammal from which the blood sample was taken. Itis also contemplated that the cells may be administered to a differentmammal. The cells may be administered to the mammal in a single dose ormultiple doses. When multiple doses are administered, the doses may beseparated from one another by, for example, one week to one month.Examples of doses of ex vivo expanded APCs include between 10⁴ and 10¹⁰cells, such as between 10⁶ cells and 10⁹ cells. As mentioned in Example5, one or more cytokines may also be administered before, during, orafter administration of the cells. It is to be understood that for anyparticular subject, specific dosage regimes should be adjusted over timeaccording to the individual need and the professional judgement of theperson administering or supervising the administration of thecompositions. Additionally, the APCs may be re-introduced as resting oractivated cells, depending on the application. Resting cells wouldrequire in vivo activation.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, and patent application wasspecifically and individually indicated to be incorporated by reference.

1. A method of preventing, stabilizing, or treating a disease, disorder,or infection in a mammal, said method comprising administering to saidmammal an anti-CD1 antibody or antibody fragment in an amount sufficientto prevent, stabilize, or treat said disease, disorder, or infection;wherein said administering is at a dosage level that allows retention ofat least 50% of the activity of CD1-reactive T cells in said mammalrelative to an untreated mammal, and wherein said disease, disorder, orinfection is an autoimmune disease, viral infection, bacterialinfection, parasitic infection, infection by a eukaryotic pathogen,allergy, asthma, inflammatory condition, graft versus host disease,graft rejection, immunodeficiency disease, spontaneous abortion,pregnancy, or cancer.
 2. A method of preventing, stabilizing, ortreating a disease, disorder, or infection in a non-rodent mammal, saidmethod comprising administering to said mammal an anti-CD1 antibody orantibody fragment in an amount sufficient to prevent, stabilize, ortreat said disease, disorder, or infection; wherein said disease,disorder, or infection is an autoimmune disease, viral infection,bacterial infection, parasitic infection, infection by a eukaryoticpathogen, allergy, asthma, inflammatory condition, graft versus hostdisease, graft rejection, immunodeficiency disease, spontaneousabortion, pregnancy, or cancer.
 3. A method of preventing, stabilizing,or treating a disease, disorder, or infection in a mammal, said methodcomprising administering to said mammal an anti-CD1 antibody or antibodyfragment in an amount sufficient to prevent, stabilize, or treat preventsaid disease, disorder, or infection; wherein said administrationincreases the amount of TGF-β or IL-10 and/or decreases the amount ofIL-12, TNF, and/or IFN-γ and wherein said disease, disorder, orinfection is an autoimmune disease, viral infection, bacterialinfection, parasitic infection, infection by a eukaryotic pathogen,allergy, asthma, inflammatory condition, graft versus host disease,graft rejection, immunodeficiency disease, spontaneous abortion,pregnancy, or cancer.
 4. A method of preventing, stabilizing, ortreating a disease, disorder, or infection in a mammal, said methodcomprising administering to said mammal an anti-CD1 antibody or antibodyfragment in an amount sufficient to prevent, stabilize, or treat saiddisease, disorder, or infection; wherein said disease, disorder, orinfection is an autoimmune disease other than lupus, viral infection,bacterial infection other than a listeria infection, parasiticinfection, infection by a eukaryotic pathogen, allergy, asthma,inflammatory condition, graft versus host disease, graft rejection,immunodeficiency disease, spontaneous abortion, pregnancy, or cancer. 5.A method of preventing, stabilizing, or treating antigen presenting cell(APC) pathogenesis in a mammal, said method comprising administering tosaid mammal an anti-CD1 antibody or antibody fragment in an amountsufficient to reduce the number or activity of at least one APC.
 6. Themethod of claim 5, wherein said antibody or antibody fragment iscovalently linked to a toxin, a radiolabel, or a molecule which targetshost defensive or catabolic processes toward said APC.
 7. The method ofclaim 5, wherein said APC pathogenesis is a Hepatitis infection,picornarirus infection, polio infection, or coxsacchie infection.
 8. Amethod of preventing, stabilizing, or treating a disease, disorder, orinfection in a mammal, said method comprising the steps of: (a)contacting a CD1-expressing APC with an anti-CD1 antibody or antibodyfragment; and (b) administering said contacted APC to said mammal in anamount sufficient to prevent, stabilize, or treat said disease,disorder, or infection; wherein said disease, disorder, or infection isan autoimmune disease, viral infection, bacterial infection, parasiticinfection, infection by a eukaryotic pathogen, allergy, asthma,inflammatory condition, graft versus host disease, graft rejection,immunodeficiency disease, spontaneous abortion, pregnancy, or cancer. 9.The method of claim 8, wherein said APC is from said mammal.
 10. Themethod of any one of claims 1-4, wherein said autoimmune disease isdiabetes, rheumatoid arthritis, pemphigus vulgaris, multiple sclerosis,or myasthenia gravis.
 11. A method of preventing, stabilizing, ortreating an infection in a mammal, said method comprising administeringto said mammal an antigen from an infectious agent and an anti-CD1antibody or antibody fragment in an amount sufficient to prevent,stabilize, or treat said infection.
 12. The method of any one of claims1-4 and 11, wherein said viral infection is a Hepatitis, picornavirus,polio, HIV, or coxsacchie infection.
 13. The method of any one of claims1-5, 8, and 11, wherein said administration is oral, intramuscular,intravenous, intraarticular, intralesional, subcutaneous,intraperitoneal, or intralesional.
 14. The method of any one of claims1-5, and 11, wherein said antibody is administered with apharmaceutically suitable carrier.
 15. The method of any one of claims1-5, 8, and 11, wherein said mammal is a human.
 16. The method any oneof claims 1-5, 8, and 11, further comprising administering one or morecytokines to said mammal.
 17. The method of claim 16, wherein saidcytokine is selected from the group consisting of IL-2, IL-4, IL-7,IL-10, IL-12, IL-13, IL-15, IL-18, IFN-α/β, IFN-γ, and GM-CSF.
 18. Themethod of claim 16, wherein said cytokines alters the ratio ofTh1/Th2/immune deviation response of T cells in said mammal.
 19. Apharmaceutical composition comprising an anti-CD1 antibody or antibodyfragment and a vaccine, an adjuvant, or an antigen from an infectiousagent.
 20. The composition of claim 19, wherein said agent is abacteria, virus, or yeast.
 21. A method for increasing the activity ornumber of an antigen-presenting cells (APC), said method comprisingadministering to said APC one or more anti-CD1 antibodies or antibodyfragments in an amount sufficient to increase the production orsecretion of a cytokine by said APC or to increase the proliferation ofsaid APC.
 22. The method of claim 21, wherein the secretion of acytokine increases by at least 50%.
 23. The method of claim 21, whereinsaid cytokine is IL-2, IL-4, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18,IFN-α/β, IFN-γ, or GM-CSF.
 24. The method of claim 21, wherein said APCis within or from a mammal diagnosed with or at increased risk for anautoimmune disease, viral infection, bacterial infection, parasiticinfection, infection by a eukaryotic pathogen, allergy, asthma,inflammatory condition, graft versus host disease, graft rejection,immunodeficiency disease, spontaneous abortion, pregnancy, or cancer.